来自Yeosuana marina sp. JLT21内切型海藻酸裂解酶的异源表达及酶学表征

doi:10.13560/j.cnki.biotech.bull.1985.2021-0539

摘要/Abstract

摘要：

褐藻寡糖有着丰富的生物学功能,酶法制备功能性褐藻寡糖具有重要实践应用价值。为发掘高活性及稳定性的褐藻寡糖制备酶,对浅海热液嗜热菌Yeosuana marina sp. JLT21中的海藻酸裂解酶YMA-1的基因在大肠杆菌中进行表达、纯化及酶活鉴定。结果发现YMA-1由306个氨基酸残基构成,是多糖裂解酶家族7（PL7）新成员;重组YMA-1酶的最适催化条件是55℃,pH 9.0,比活力1.3×10⁴U/mg,Cu²⁺ 可有效促进酶活;在37℃,pH 9.0 条件下,该酶对海藻酸钠、聚甘露糖醛酸和聚古罗糖醛酸的比活力分别达到（5 201.21±86.46）U/mg、（6 399.73±253.12）U/mg和（3 751.68±116.25）U/mg,酶解海藻酸钠终产物多为不饱和三糖和四糖,表现出内切双功能型海藻酸裂解酶活性。YMA-1酶作为PL7家族中较宽底物谱、高活性及稳定性的内切海藻酸裂解酶,在高效绿色生产功能性褐藻寡糖上有着潜在应用价值。

关键词: 内切型海藻酸裂解酶, 重组表达, 酶学性质, Yeosuana marina sp. JLT21

Abstract:

Alginate oligosaccharides（AO）have rich biological activity,preparing AO using enzymatic approach is of important,practical and applicable value. To mine highly active and stable enzymes for preparing AO,a gene from an alginate lyase YAM-1 of Yeosuana marina sp. JLT21 was expressed in Escherichia coli,and the enzyme was purified and characterized. As one of polysaccharide lyase family 7（PL7）members,YMA-1 consisted of 306 amino acids. The recombinant YMA-1 showed the optimal activity of 1.3×10⁴ U/mg at pH 9.0 and 55℃,and Cu²⁺ efficiently stimulated its activity. YMA-1 also demonstrated a highly catalytic activity towards sodium alginate,poly G and poly M,and their specific activities were（5 201.21±86.46）,（6 399.73±253.12）and（3 751.68±116.25）U/mg,respectively under 37℃ and pH 9.0. The end-products from the enzymolysis of sodium alginate were mainly unsaturated trisaccharides and tetrasaccharides. Duo to its broad substrate spectrum,high activity and thermostability,YMA-1 as endo-type alginate lyase in PL7 family has potential applications in green and efficient production of alginate oligosaccharides.

Key words: endo-type alginate lyase, recombinant expression, enzymatic properties, Yeosuana marina sp. JLT21

常晴, 束月蓉, 王文韬, 蒋昊, 延泉德, 钱政, 高雪纯, 吴金鸿, 张勇. 来自Yeosuana marina sp. JLT21内切型海藻酸裂解酶的异源表达及酶学表征[J]. 生物技术通报, 2022, 38(2): 123-131.

CHANG Qing, SHU Yue-rong, WANG Wen-tao, JIANG Hao, YAN Quan-de, QIAN Zheng, GAO Xue-chun, WU Jin-hong, ZHANG Yong. Heterologous Expression and Characterization of Endo-type Alginate Lyase from Yeosuana marina sp. JLT21[J]. Biotechnology Bulletin, 2022, 38(2): 123-131.

图/表 6

图1 海藻酸裂解酶YMA-1的系统进化树及PL7家族酶氨基酸序列比对分析 A：Bootstrap 是从1 000个重复中获得的置信度值（以百分比表示）,分支的右侧是海藻酸分解菌的种类和在 GenBank 上的登录号,标尺表示 20%的序列差异;B：FlAlyA来自 Flavobacterium sp. UMI-01（BAP05660）,A1-II’ 来自 Sphingomonas sp. A1（BAD16656）,AlyA来自Klebsiella pneumoniae（AAA25049）,AlyA5来自 Zobellia galactanivorans DsijT（CAZ98266.1）,AlyA1来自 Z. galactanivorans DsiJT（CAZ95239）,AlgAT5来自 Defluviitalea phaphyphila（WP_058486006.1）,PA1167 来自 Pseudomonas aeruginosa PAO1（AAG04556）,AlyC3来自 Psychromonas sp.（QOP59290.1）

Fig.1 Phylogenetic tree of alginate lyase YMA-1 and amino acid sequence alignment analysis of PL7 family enzyme A：Bootstraps are the confidence values obtained from 1,000 replications（%）. Species of alginolytic bacteria and the accession number from GenBank are listed on the right. Scale bar indicates approximately 20% sequence difference. B：FlAlyA from Flavobacterium sp. UMI-01（BAP05660）,A1-II’ from Sphingomonas sp. A1（BAD16656）,AlyA from Klebsiella pneumoniae（AAA25049）,AlyA5 from Zobellia galactanivorans DsijT（CAZ98266.1）,AlyA1 from Z. galactanivorans DsiJT（CAZ95239）,AlgAT5 from Defluviitalea phaphyphila（WP_058486006.1）,PA1167 from Pseudomonas aeruginosa PAO1（AAG04556）,and AlyC3 from Psychromonas sp.（QOP59290.1）

图2 海藻酸裂解酶YMA-1的重组表达及纯化分析 A：SDS-PAGE检测重组酶YMA-1纯度,M：蛋白 Marker;1：粗酶液,2：20 mmol/L 咪唑流穿液,3：30 mmol/L咪唑流穿液,4：50 mmol/L咪唑流穿液,5：80 mmol/L咪唑流穿液,6：纯化后的重组酶YMA-1;B：重组酶YMA-1的分子筛层析色谱图

Fig.2 Recombinant expression,purification and analysis of alginate lyase YMA-1 A：Detecting the purity of recombinase YMA-1 via SDS-PAGE. M：Protein marker. 1：Crude enzyme. 2：Flow-through with 20 mmol/L imidazole buffer;3：Flow-through with 30 mM imidazole buffer. 4：Flow-through with 50 mmol/L imidazole buffer. 5：Flow-through with 80 mmol/L imidazole buffer. 6：Purified recombinant enzyme YMA-1. B：Gel filtration chromatography analysis of recombinant enzyme YMA-1

图3 海藻酸裂解酶YMA-1在不同pH、温度和金属离子条件下活性分析 A：重组酶YMA-1在 pH在5.0-10.0条件下活性分析,YMA-1催化最适pH 9.0,在该条件下酶比活力可达4.7×103 U/mg;B：重组酶YMA-1在10-60℃ 度下活性分析,最适温度为55℃,酶比活力高达1.3×104 U/mg;C：差示扫描量热法（DSC）测得重组酶YMA-1 的Tm值37℃;D：不同金属离子对重组酶YMA-1活性的影响分析

Fig.3 Activities of alginate lyase YMA-1 under different pH,temperature and metal ions A：Determination of YMA-1 activity at pH 5.0 to 10.0. YMA-1 catalysis was optimal at pH 9.0,and its specific activity of the enzyme was 4.7×103 U/mg. B：Examination of YMA-1 activity at 10℃ to 60℃. YMA-1 showed the optimal activity of 1.3×104 U/mg at 55℃. C：Measurement of YMA-1 thermostability Tm value was determined by differential scanning calorimetry（DSC）. D：Effects of various metal ions on the activity of YMA-1

图4 海藻酸裂解酶YMA-1的底物偏好性分析 A：海藻酸裂解酶催化底物模式;根据海藻酸裂解酶作用的底物结构不同,可以分为 Poly M型裂解酶、Poly G型裂解酶和双功能型裂解酶;B：重组YMA-1酶对3种不同结构海藻酸底物偏好性分析;该酶可对3种不同结构的海藻酸底物表现出高活性,证实了其是一种双功能海藻酸裂解酶

Fig.4 Substrate preference of alginate lyase YMA-1 A：Substrate specificity of alginate lyases. Alginate lyases can be classified into Poly M lyase and Poly G lyase and bi-functional lyase according to chemical structures of substrates acting with alginate lyase. B：Preference of YMA-1 on 3 substrates in different structures,it presents high activity on 3 alginic acid substrates in different structure,confirming it is a bi-functional alginate lyase

图5 海藻酸裂解酶YMA-1催化酶动力学米氏图

Fig.5 Michaelis-Menten kinetics of alginate lyase YMA-1

图6 海藻酸裂解酶YMA-1催化海藻酸钠降解的寡糖产物分析 A：海藻酸钠降解产物HPLC分析图,YMA-1降解海藻酸钠通过HPLC共检测到4种不同产物,出峰时间分别为3.92,4.04,4.13和4.23 min;B：海藻酸钠降解产物ESI-MS鉴定图谱,重组酶降解海藻酸钠最终产物对应时间分别为不饱和二糖（ΔDP2）（[M-H]-=351.0）,不饱和三糖（ΔDP3）（[M-H]-=527.09）,不饱和四糖（ΔDP4）（[M-H]-=703.12）和不饱和五糖（ΔDP5）（[M-H]-=879.15）

Fig.6 LC-MS analysis of the oligosaccharide products from degrading sodium alginates by YMA-1 catalysis A：HPLC analysis of products of YMA-1 catalysis. Four different end-products were detected,and the peak times were 3.92,4.04,4.13 and 4.23 min,respectively. B：ESI-MST analysis of products of YMA-1 catalysis. The end-products were unsaturated disaccharides（ΔDP2）（[M-H]-=351.0）,unsaturated trisaccharides（ΔDP3）（[M-H]-=527.09）,unsaturated tetrasaccharide（ΔDP4）（[M-H]-=703.12）and unsaturated pentasaccharide（ΔDP5）（[M-H]-=879.15）,respectively

参考文献 20

[1]	Kloareg B, Demarty M, Mabeau S. Ion-exchange properties of isolated cell walls of brown algae:the interstitial solution[J]. J Exp Bot, 1987, 38(10):1652-1662. doi: 10.1093/jxb/38.10.1652 URL
[2]	Sellimi S, Younes I, Ayed HB, et al. Structural, physicochemical and antioxidant properties of sodium alginate isolated from a Tunisian brown seaweed[J]. Int J Biol Macromol, 2015, 72:1358-1367. doi: 10.1016/j.ijbiomac.2014.10.016 URL
[3]	Tang JC, Taniguchi H, Chu H, et al. Isolation and characterization of alginate-degrading bacteria for disposal of seaweed wastes[J]. Lett Appl Microbiol, 2009, 48(1):38-43. doi: 10.1111/j.1472-765X.2008.02481.x pmid: 19018967
[4]	Wong TY, Preston LA, Schiller NL. Alginate lyase:review of major sources and enzyme characteristics, structure-function analysis, biological roles, and applications[J]. Annu Rev Microbiol, 2000, 54(1):289-340. doi: 10.1146/micro.2000.54.issue-1 URL
[5]	Jain S, Ohman DE. Role of an alginate lyase for alginate transport in mucoid Pseudomonas aeruginosa[J]. Infect Immun, 2005, 73(10):6429-6436. doi: 10.1128/IAI.73.10.6429-6436.2005 URL
[6]	Cheng D, Jiang C, Xu J, et al. Characteristics and applications of alginate lyases:a review[J]. Int J Biol Macromol, 2020, 164:1304-1320. doi: 10.1016/j.ijbiomac.2020.07.199 URL
[7]	Zhu B, Yin H. Alginate lyase:Review of major sources and classification, properties, structure-function analysis and applications[J]. Bioengineered, 2015, 6(3):125-131. doi: 10.1080/21655979.2015.1030543 URL
[8]	Xu F, Wang P, Zhang YZ, et al. Diversity of three-dimensional structures and catalytic mechanisms of alginate lyases[J]. Appl Environ Microbiol, 2018, 84(3):e02040-17.
[9]	Lombard V, Bernard T, Rancurel C, et al. A hierarchical classification of polysaccharide lyases for glycogenomics[J]. Biochem J, 2010, 432(3):437-444. doi: 10.1042/BJ20101185 URL
[10]	Wang B, Ji SQ, Lu M, et al. Biochemical and structural characterization of alginate lyases:an update[J]. Curr Biotechnol, 2015, 4(3):223-239. doi: 10.2174/2211550104666150723231423 URL
[11]	Nakata S, Murata K, Hashimoto W, et al. Uncovering the reactive nature of 4-deoxy- l - erythro -5-hexoseulose uronate for the utilization of alginate, a promising marine biopolymer[J]. Sci Rep, 2019, 9:17147. doi: 10.1038/s41598-019-53597-1 pmid: 31748627
[12]	Kim HS, Lee CG, Lee EY. Alginate lyase:Structure, property, and application[J]. Biotechnol Bioprocess Eng, 2011, 16(5):843-851. doi: 10.1007/s12257-011-0352-8 URL
[13]	Wang Y, Han F, Hu B, et al. In vivo prebiotic properties of alginate oligosaccharides prepared through enzymatic hydrolysis of alginate[J]. Nutr Res, 2006, 26(11):597-603. doi: 10.1016/j.nutres.2006.09.015 URL
[14]	Islan GA, Bosio VE, Castro GR. Alginate lyase and ciprofloxacin co-immobilization on biopolymeric microspheres for cystic fibrosis treatment[J]. Macromol Biosci, 2013, 13(9):1238-1248. doi: 10.1002/mabi.201300134 URL
[15]	Tang L, Wang Y, Gao S, et al. Biochemical characteristics and molecular mechanism of an exo-type alginate lyase V_xAly7D and its use for the preparation of unsaturated monosaccharides[J]. Biotechnol Biofuels, 2020, 13:99. doi: 10.1186/s13068-020-01738-4 URL
[16]	Lu D, Zhang Q, Wang S, et al. Biochemical characteristics and synergistic effect of two novel alginate lyases from Photobacterium sp. FC615[J]. Biotechnol Biofuels, 2019, 12:260. doi: 10.1186/s13068-019-1600-y URL
[17]	Yamasaki M, Moriwaki S, Miyake O, et al. Structure and function of a hypothetical Pseudomonas aeruginosa protein PA1167 classified into family PL-7:a novel alginate lyase with a beta-sandwich fold[J]. J Biol Chem, 2004, 279(30):31863-31872. doi: 10.1074/jbc.M402466200 URL
[18]	Kobayashi T, Uchimura K, Miyazaki M, et al. A new high-alkaline alginate lyase from a deep-sea bacterium Agarivorans sp[J]. Extremophiles, 2009, 13(1):121-129. doi: 10.1007/s00792-008-0201-7 pmid: 19002649
[19]	Inoue A, Anraku M, Nakagawa S, et al. Discovery of a novel alginate lyase from Nitratiruptor sp. SB155-2 thriving at deep-sea hydrothermal vents and identification of the residues responsible for its heat stability[J]. J Biol Chem, 2016, 291(30):15551-15563. doi: 10.1074/jbc.M115.713230 URL
[20]	Li S, Wang L, Chen X, et al. Cloning, expression, and biochemical characterization of two new oligoalginate lyases with synergistic degradation capability[J]. Mar Biotechnol:NY, 2018, 20(1):75-86. doi: 10.1007/s10126-017-9788-y URL